Nesterenkonia salmonea sp. nov. and Nesterenkonia sphaerica sp. nov., isolated from the Southern Atlantic Ocean

923 Nesterenkonia salmonea sp. nov. and Nesterenkonia sphaerica sp. nov., isolated from the Southern AtlanticOcean GaiyunZhang*, LinaWang, FuquanXie, ShengxiangPei and LiJiang TAXONOMIC DESCRIPTION Zhang etal., Int. J. Syst. Evol. Microbiol. 2020;70:923–928 DOI 10.1099/ijsem.0.003847 Author affiliations: 1Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, Fujian, PR China. *Correspondence: Gaiyun Zhang, zhgyun@ tio. org. cn Keywords: Nesterenkonia salmonea sp. nov.; Nesterenkonia sphaerica sp. nov.; polyphasic taxonomy; marine sediment. Abbreviations: ANI, average nucleotide identity; DDH, DNA–DNA hybridization; DPG, diphosphatidylglycerol; GL, unidentified glycolipid; L, unidentified lipid; MA, marine agar; MB, marine broth; ML, maximumlikelihood; MP, maximumparsimony; NJ, neighbourjoining; PG, phosphatidylglycerol; PI, phosphatidylinositol; PL, unidentified phospholipid; UPLCMS, ultra performance liquid chromatographymass spectrometry. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains GY074T and GY239T are KT751085 and KT751084, respectively. GenBank/EMBL/DDBJ accession numbers for the draft genome sequences of strains GY074T and GY239T are VAVZ00000000 and VAWA00000000, respectively. One supplementary table and two supplementary figures are available with the online version of this article. 003847 © 2020 The Authors Abstract Two Grampositive, aerobic, nonmotile and nonsporeforming actinobacteria, strains GY074T and GY239T, were isolated from deepsea sediment of the Southern Atlantic Ocean. The results of phylogenetic analysis of 16S rRNA gene sequences placed both isolates within the genus Nesterenkonia, and showed a sequence similarity of 98.3 % between the two strains and similarites of 94.3–97.2 % with respect to Nesterenkonia species with validly published names. Based on wholegenome sequences, the values of in silico DNA–DNA hybridization and the average nucleotide identity between strains GY074T and GY239T were 21.2 and 78.1 %, respectively, less than the proposed cutoff level for species delineation, i.e. 70 and 95 %. For both strains, the major cellular fatty acids were anteisoC17 : 0 and anteisoC15 : 0, and the major menaquinones were MK-7, MK-8 and MK-9. The major polar lipid contents of the two strains were similar with phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid. The genomic DNA G+C contents of strains GY074T and GY239T were 61.1 and 64.2 mol%, respectively. On the basis of the phylogenetic analysis and physiological and chemotaxonomic data, the isolates represent two novel species of the genus Nesterenkonia, for which the names Nesterenkonia salmonea sp. nov. (type strain GY074T=KCTC 39639T=MCCC 1A11256T) and Nesterenkonia sphaerica sp. nov. (type strain GY239T=KCTC 39640T=MCCC 1A10688T) are proposed. The genus Nesterenkonia, was first proposed by Stackebrandt et al. [1], and emended subsequently by Collins et al. [2] and Li et al. [3]. At the time of writing, the genus Nesterenkonia comprised 20 species with validly published names, of which Nesterenkonia endophytica was recently described [4]. These species have been isolated from various environments including saline and alkaline soils or lakes [1, 2, 5–7], paper and cotton pulp mill effluents [8, 9], fermented seafoods [10], plant tissues [4, 11] and the faeces of a woman patient [12] et al. Members of the genus Nesterenkonia are Grampositive, nonsporeforming, rodor coccishaped, aerobic, moderately halophilic or halotolerant [1–3]. Nesterenkonia species have diphosphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as major polar lipids, isoand anteisobranched fatty acids with 15–17 carbons as dominated cellular fatty acids, and A4αtype peptidoglycan with lLys–Gly–lGlu, lLys–lGlu or Lys–Gly–dAsp [1–3]. Prior to this study, except for Nesterenkonia alkaliphile [13], none of the above Nesterenkonia species were isolated from marine sediments. In this study, two novel Nesterenkonia strains, GY074 T and GY239T, were isolated from sediment samples of the Southern Atlantic Ocean. Based on the results of polyphasic analyses, we propose that the isolates represent two novel species in the genus Nesterenkonia. Strains GY074T and GY239T were isolated from different sediment samples collected from the Southern Atlantic Ocean at sites MC1 (−3223 m, 12.85° W 18.48° S) and MC2 (−2859 m, 13.41° W 15.18° S), respectively. Both strains were isolated using the dispersion and differential centrifugation technique [14] on vitamin agar [B vitamins (0.5 mg each of thiamine–HCl, riboflavin, niacin, pyridoxin–HC1, Ca– pantothenate, inositol, paminobenzoic acid, and 0.25 mg biotin), 18 g agar, 1000 ml filtered seawater and pH 7.2±0.1] at 30 °C. The isolates were routinely cultured on marine agar 2216 (MA; Difco) and maintained at −80 °C as suspensions 924 Zhang etal., Int. J. Syst. Evol. Microbiol. 2020;70:923–928 in marine broth 2216 (MB; Difco) containing glycerol (20 %, w/v). Morphological and physiological tests of strains GY074T and GY239T were performed as follows. Cell morphology was observed with light microscope (BH-2, Olympus) and transmission electron microscopy (H-600, Hitachi) using cells that had been grown for 24–36 h on MA medium at 30 °C. Gram staining was carried out by the standard Gram's reaction as described by Cerny [15]. Growth at different temperatures, salinities and pHs were assessed using methods described by Yin et al. [16]. Cell motility was confirmed by the development of turbidity throughout a tube containing semisolid medium [17]. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber on MA at 30 °C for about 1 month. Catalase activity was determined by bubble production on addition of a drop of 3 % (v/v) H2O2 and oxidase activity was determined using 1 % (w/v) tetramethylpphenylenediamine. Nitrate reduction, methyl red test, production of H2S and melanin, Voges–Proskauer reaction, and hydrolysis of starch, cellulose, gelatin and casein were determined as described by Ruan and Huang [18]. Other physiological and biochemical characteristics were determined using the API 20NE and API ZYM systems (bioMérieux) according to the instructions of the manufacturer. Strain GY074T formed darksalmon, circular, convex, smooth and opaque colonies with entire margins, approximately 1.0 mm in diameter after 6 days at 30 °C. The cells were Grampositive, nonmotile, obligate aerobic rodshaped, 0.4–0.5 µm wide and 0.7–1.0 µm long (Fig. S1, available in the online version of this article). Strains GY239T and GY074T had similar morphological characteristics except the cell shape and colour of colonies. Strains GY239T produced lightyellow pigments and cells were coccoidshaped with 0.7–0.8 µm in diameter (Fig. S1). The growth temperature range for strain GY074T was 4–40 °C, while that for strain GY239T was 10–40 °C. Strain GY074T was negative for oxidase and grew in the presence of 10 % NaCl, whereas GY239T was positive for oxidase and grew in the presence of up to 9 % NaCl. These data indicated that the two isolates were different physiologically. The detailed results of the physiological characterizations are given in the species descriptions; features that serve to distinguish between the two strains and related Nesterenkonia species are listed in Table1. Table 1. Differential morphological, physiological and chemical characteristics of strains GY074T, GY239T and their phylogenetically closest relatives Strains: 1, GY074T; 2, GY239T; 3, Nesterenkonia halophila YIM 70179T; 4, Nesterenkonia halobia JCM11483T; 5, Nesterenkonia suensis DSM22748T; 6, Nesterenkonia lutea YIM 70081T. All data are from this study except for major menaquinones, peptidoglycan type, polar lipids and the DNA G+C contents of strains 3–6, which are from Li et al. [38], Stackebrandt et al. [1], Govender et al. [5] and Li et al. [3], respectively. +, Positive; −, negative. Characteristic 1 2 3 4 5 6 Cell shape Rod Cocci Cocci Cocci Rod Cocci Colony pigmentation (MA medium) Dark salmon Light yellow White White Yellow Yellow Nitrate reduction − − + − + + Activities of: βGalactosidase − − + + − + αGlucosidase − − + + + + βGlucosidase − − + + + + Arginine dihydrolase − − + + + − Oxidase − + − + − − Hydrolysis of: Starch − − − + + − Gelatin − − + − + − Major menaquinones MK-7, MK-8, MK-9 MK-7, MK-8, MK-9 MK-8, MK-9, MK-7 MK-8, MK-9 MK-7, MK-8, MK-9 MK −7, MK-8 Peptidoglycan type lLys–GlylGlu lLys–lGlu lLys–GlylGlu lLys–GlylGlu lLys–Gly–dAsp Lys–Gly– dAsp Polar lipids PI, PG, DPG, GL, L, PL PI, PG, DPG, GL, 2PLs, 3Ls PI, PG, DPG, GL PG, DPG, PI, GL PG, DPG, PI, GL PG, DPG, PI, GL DNA G+C content (mol%) 61.1 64.3 68.4 71.5 64.8 64.5 925 Zhang etal., Int. J. Syst. Evol. Microbiol. 2020;70:923–928 Genomic DNA extraction and PCR amplification of the 16S rRNA gene were performed as described previously [19]. The nearcomplete 16S rRNA gene sequences were obtained with primers 27F (5′- AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′- GGTTACCTTGTTACGACTT-3′) [20]. The sequence was compared with 16S rRNA gene sequences of valid species from GenBank via the blast program and the EzTaxone server [21]. Evolutionary analyses were conducted in mega7 [22]. Phylogenetic trees were reconstructed using three treemaking algorithms: maximumparsimony (MP) [23], maximumlikelihood (ML) [24] and neighbourjoining (NJ) [25]. All positions containing gaps and missing data were eliminated. For the ML analysis, initial tree(s) for the heuristic search were obtained automatically by applying the NeighborJoin and BioNJ algorithms to a matrix of pairwise distances estimated using the maximumcompositelikelihood approach, and then selecting the topology with superior log likelihood value. The ML tree was built using the Tamura–Nei model with the highest log likelihood (−4264.8503) [26]. The MP tree was obtained using the SubtreePruningRegrafting algorithm (p. 126 in [27].) with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). The NJ tree was built using the maximumcompositelikelihood method. The stability of relationships was evaluated by performing a bootstrap analysis based on 1000 resamplings. The almostcomplete 16S rRNA gene sequences for strains GY074T (1491 bp, GenBank accession number KT751085) and GY239T (1489 bp, GenBank accession number KT751084) were obtained. Strain GY074T showed the highest 16S rRNA gene sequence similarity to strain GY239T (98.3 %), followed by the type strains of Nesterenkonia lutea (97.2 %), Nesterenkonia suensis (97.1 %) and Nesterenkonia halobia (97.1 %). Strain GY239T showed the closest relationships with strain GY074T (98.3 %) and the type strain of N. halophila (97.1 %). Lower level sequence similarity values (≤97.0 %) of the two isolates were found to other species from the genus Nesterenkonia. The topologies in the phylogenetic tree using three treemaking algorithms showed that strain GY074T constituted a separated branch with strain GY239T in the clade of the genus Nesterenkonia at a high bootstrap value 98 % (Fig.1). Thus, it was clear that these two isolates belonged to the genus Nesterenkonia but were different from these validly published Nesterenkonia species. To further clarify the taxonomic relationship of strains GY074T and GY239T, the genome sequences were obtained using an Illumina HiSeq X Ten system at the Shanghai Majorbio BioPharm Technology Co., Ltd (PR China). The pairedend libraries were constructed from sheared genomic DNA consisting of 400–500 bp fragments, and sequencing was performed according to the manufacturer's instructions. The reads were assembled using SOAPdenovo version 2.04 Nesterenkonia jeotgali JG-241T (AY928901) Nesterenkonia sandarakina YIM 70009T (AY588277) Nesterenkonia halotolerans YIM 70084T (AY226508) Nesterenkonia aurantiaca DSM 27373T (HG795012) Nesterenkonia lutea YIM 70081T (AY588278) Nesterenkonia sphaerica GY239T (KT751084) Nesterenkonia salmonea GY074T (KT751085) Nesterenkonia halophila YIM 70179T (AY820953) Nesterenkonia halobia DSM 20541T (X80747) Nesterenkonia aethiopica DSM 17733T (AY574575) Nesterenkonia suensis Sua-BAC020T (FJ948172) Nesterenkonia xinjiangensis YIM 70097T (AY226510) Nesterenkonia lacusekhoensis IFAM EL-30T (AJ290397) Nesterenkonia cremea 10CT (LT601575) Nesterenkonia flava CAAS 251T (EF680886) Nesterenkonia alkaliphila F10T (KF914157) Nesterenkonia alba DSM 19423T (ATXP01000006) Nesterenkonia populi GP10-3T (KP057085) Nesterenkonia pannonica BV-35T (LN610504) Nesterenkonia endophyticus EGI 60016T (MF630917) Nesterenkonia rhizosphaerae EGI 80099T (KF040423) Nesterenkonia massiliensis NP1T (JX424770) Arthrobacter nasiphocae CCUG 42953 T (AJ292364) 100 100 75 92 79 100 98 100 99 70 74 97 85 69 55 0.005 Fig. 1. Phylogenetic analysis of strains GY074T and GY239T and closely related members of the genus Nesterenkonia based on 16S rRNA gene sequences. GenBank accession numbers are given in parentheses. Multiple alignments, distance calculations and clustering with the neighbourjoining, maximumlikelihood and maximumparsimony methods were performed by using the software package mega version 7. Bootstrap values based on 1000 replications are shown as percentages at the branching points. Bar, 0.005 nucleotide substitutions per nucleotide position. Filled circles (●) indicate that these branches cluster similarly using the different test algorithms. 926 Zhang etal., Int. J. Syst. Evol. Microbiol. 2020;70:923–928 (http:// soap. genomics. org. cn/). The genome sizes of strains GY074 T (GenBank accession number VAVZ00000000) and GY239T (GenBank accession number VAWA00000000) were 3 283 708 bp and 2 791 466 bp long and the genome DNA G+C contents were 61.1 and 64.3 mol%, respectively. Average nucleotide identity (ANI) was calculated by using the OrthoANIu algorithm [28]. The DNA–DNA hybridization (DDH) value between strains was calculated in silico with the GenometoGenome Distance Calculator (http:// ggdc. dsmz. de/) [29]. The ANI value between strains GY074T and GY239T was 78.1 %, which is below the proposed threshold of 95–96 % [28]. Meanwhile, in silico DDH values of the two isolates was 21.2 %, which is also less than the 70 % proposed threshold for the same species designation [30], supporting the conclusion that strains GY074 T and GY239 T were two novel species of the genus Nesterenkonia. For chemotaxonomic analyses, biomass was obtained from cultures grown in shake flasks containing MB and incubated at 30 °C for 3 days. Polar lipids were extracted using the integrated procedure of Minnikin et al. [31], separated by two dimensional TLC and identified using the procedure described by Minnikin et al. [32]. Respiratory quinones were analysed using UPLCMS as described by Kaiser et al. [33]. For cellular fatty acid analysis, fatty acid methyl esters were prepared and analysed according to the midi standard protocol (Sherlock Microbial Identification System, version 6.0) and identified by using the TSBA6.0 database of the Microbial Identification System [34]. Cellwall peptidoglycan was prepared and analysed using methods described by Schleifer and Kandler [35], Schleifer [36] and Schumann [37]. Chemotaxonomically, the dominant fatty acids (>10 % of the total fatty acids) were anteisoC17 : 0 (32.7 %), anteisoC15 : 0 (20.8 %) and anteisoC17 : 1 (11.5 %) for strain GY074T, and anteisoC15 : 0 (37.9 %) and anteisoC17 : 0 (26.3 %) for strain GY239T. AnteisoC 15 : 0 and anteisoC 17 : 0 were typically found as major components in members of the genus Nesterenkonia [1, 38]. Significant differences in the proportions of several fatty acids were found between the isolates and the reference strains. Detailed fatty acid profiles of the isolates and the reference strains are presented in Table S1. The peptidoglycans of strains GY074T and GY239T were of type A4α with lLysGlylGlu and lLys–lGlu, respectively, which were also found in some members of the genus Nesterenkonia [2, 6]. The major isoprenoid quinones were MK-7, MK-8 and MK-9 in the ratios 25.2 : 56.3 : 18.5 (GY074T) and 9.7 : 83.1 : 7.2 (GY239 T ). The major polar lipid contents of the two strains were similar with phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid. In addition, one unidentified phospholipid and one unidentified lipid were determined for strain GY074T, meanwhile, two unidentified phospholipids and three unidentified lipids were determined for strain GY239T (Fig. S2). In summary, based on their morphological, physiological and chemotaxonomic characteristics, strains GY074T and GY239T represent two novel species within the genus Nesterenkonia, for which the names Nesterenkonia salmonea sp. nov. and Nesterenkonia sphaerica sp. nov. are proposed. DESCRIPTION Of NestereNkoNia salmoNea SP. NOv. Nesterenkonia salmonea ( sal. mo'ne.a. N.L. fem. adj. salmonea darksalmoncoloured, referring to the colour of the colonies on solid medium). Cells are Gramstainpositive, aerobic, nonmotile and rodshaped, approximately 0.4–0.5 µm in diameter and 0.7–1.0 µm long. Colonies on MA are approximately 1 mm in diameter, opaque, smooth, circular with regular margins, convex and dark salmon in colour after 6 days at 30 °C. Growth occurs in 0–10 % (w/v) NaCl (optimum, 1–4 %), at pH 7–12 (pH 9–10) and at 4–40 °C (30–35 °C). Catalasepositive and oxidasenegative. Negative for nitrate reduction, methyl red test, production of H2S and melanin, Voges–Proskauer reaction, and hydrolysis of starch, cellulose and casein. Assays using the API ZYM system are positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, αchymotrypsin and naphtholASBIphosphohydrolase; weakly positive for valine arylamidase, cystine arylamidase and acid phosphatase; negative for Nacetyl-βglucosaminidase, αglucosidase, lipase (C14), trypsin, αgalactosidase, βglucuronidase, βgalactosidase, βglucosidase, αmannosidase and βfucosidase. Analysis using the API 20NE system reveals that cells are positive for hydrolysis of aesculin and gelatin; negative for the remaining 18 substrates. The major menaquinones are MK-7, MK-8 and MK-9. The peptidoglycan type is A4α, lLysGly–lGlu. The polar lipids detected are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, one unidentified glycolipid, one unidentified lipid and one unidentified phospholipid. The major fatty acids are anteisoC17 : 0, anteisoC15 : 0 and anteisoC17 : 1; in addition, significant amounts of isoC15 : 0, isoC16 : 0 and isoC17 : 0 are also present. The genomic DNA G+C content of the type strain is 61.1 mol%. The type strain, GY074 T = (KCTC 39639 T =MCCC 1A11256 T ), was isolated from marine sediment collected from the Southern Atlantic Ocean. DESCRIPTION Of NestereNkoNia sphaerica SP. NOv. Nesterenkonia sphaerica (sphae' ri. ca. L. fem. adj. sphaerica spherical, referring to the cell shape of the type strain). Cells are Gramstainpositive, aerobic, nonmotile and nonsporeforming cocci (diameter 0.7–0.8 µm) without any flagella. Colonies on MA are approximately 1 mm in diameter, opaque, smooth, circular with regular margins, convex and lightyellow in colour after 6 days at 30 °C. Growth occurs in 0–9 % (w/v) NaCl (optimum, 1–4 %), at pH 7–12 (pH 9–10) and at 10–40 °C (30–35 °C). Positive for catalase and oxidase. Negative for nitrate reduction, methyl red test, production of H2S and melanin, Voges– Proskauer reaction, and hydrolysis of starch, cellulose and casein. Assays using the API ZYM system are positive for 927 Zhang etal., Int. J. Syst. Evol. Microbiol. 2020;70:923–928 alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase and naphtholASBIphosphohydrolase; weakly positive for valine arylamidase and acid phosphatase; negative for Nacetyl-βglucosaminidase, cystine arylamidase, αchymotrypsin, αglucosidase, lipase (C14), trypsin, αgalactosidase, βglucuronidase, βgalactosidase, βglucosidase, αmannosidase and βfucosidase. Analysis using the API 20NE system reveals that cells are positive for hydrolysis of aesculin and gelatin; negative for the remaining 18 substrates. The major menaquinones are MK-7, MK-8 and MK-9. The peptidoglycan type is A4α, lLys–lGlu. The polar lipids detected are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, one unidentified glycolipid, two unidentified phospholipids and three unidentified lipids. The major fatty acids are anteisoC15 : 0 and anteisoC17 : 0; in addition, significant amounts of isoC16 : 0, isoC15 : 0 and C16 : 0 are also present. The genomic DNA G+C content of the type strain is 64.3 mol%. The type strain, GY239 T =(KCTC 39640 T =MCCC 1A10688 T ), was isolated from marine sediment collected from the Southern Atlantic Ocean. Funding information This work was supported by grants from the China Ocean Mineral Resources R and D Association (COMRA) Program (No. DY135B2-01) and the Scientific Research Foundation of Third Institute of Oceanography, MNR (No. 2019011). Conflicts of interest The authors declare that there are no conflicts of interest. References 1. Stackebrandt E, Koch C, Gvozdiak O, Schumann P. 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